Abstract

Purpose: The differentiation marker 2M6
has been used to identify Müller cells within the developing chick
retina for several years, although the molecular identity of 2M6 was
not known. This study was aimed at determining the identity of the
protein antigen recognized by the 2M6 monoclonal antibody.

Methods: Affinity chromatography and
subsequent mass spectrometry were used to determine the molecular
identity of the 2M6 antigen. Immunohistochemistry of monolayer
preparations and paraffin-embedded sections of chick retina were
performed to localize expression of the 2M6 antigen within cells of the
chick retina.

Results: Mass spectrometry analyses
revealed that the 2M6 antigen is identical (with 95% probability) to
the protein known as TopAP, which is a member of the
sarcolemmal membrane-associated protein family of proteins. The 2M6
polypeptide is expressed by Müller glial cells as well as boundary
cells within the chick retina. Expression localizes to intracellular
membrane structures within those cells.

Conclusions: Members of the sarcolemmal
membrane-associated protein family of proteins have been implicated in
structural and functional roles related to the cytoskeleton and Ca+2
release from internal stores. It is thought that 2M6 plays a similar
role in Müller cells of the vertebrate retina.

Development of the vertebrate retina proceeds such that mitotic
cells leave the cell cycle and differentiate into the various cell
types found within the tissue. All retina cells differentiate from a
common progenitor cell population; cones are “born” relatively early in
development and rods and Müller glial cells are the last cell types
produced (reviewed in [1]).
The neurons and glial cells rely on topographic cues and expression of
differentiation factors to migrate to the appropriate layers of the
retina and also for guidance of projections [1]. Often these differentiation factors are used
as markers to identify cells within the developing retina.

One such differentiation marker is the 2M6 antigen, which was first
identified by Schlosshauer et al. [2]. The 2M6 antigen is a 40–46 kDa protein
expressed after major laminations of chick retinal tissue are
established [2].
Linser et al. [3]
reported the presence of a pool of mitotically active cells that have
glial-like qualities and express the 2M6 antigen. It is thought that
2M6 influences glial differentiation in the neural retina [3] and is considered a
definitive marker of Müller glia [3,4].

In 1995 Savitt et al. [5]
reported that TopAP is expressed during periods of
retinotectal synapse formation in the chick retina. In embryonic day 8
(E8) chick retina, TopAP, a 40-kDa protein, has graded
expression along the anterior to posterior axis in retina and optic
tectum [5].
Indeed, the name refers to the fact that the protein is a topographic
marker expressed along the anterior-posterior axis [5]. Hydropathy plot
analyses of the translated cDNA sequence of TopAP suggest
that it is a membrane-associated protein [5]. Savitt et al. [5] proposed that TopAP is crucial for
synapse connectivity within the developing neural retina.

Presently, we report that TopAP is the 2M6 antigen.
Affinity purification of detergent-treated chick retina lysates and
subsequent mass spectrometry (MS/MS) analysis indicate that the protein
recognized by the 2M6 antibody is identical to the protein named TopAP.
Liquid chromatography (LC) and and tandem MS/MS were performed at the
University of Florida biotechnology core facility in Gainesville, FL.
The immunohistochemical data presented herein indicate that 2M6 (TopAP)
is an intracellular protein within Müller glial cells. The 2M6 (TopAP)
protein belongs to a family of proteins associated with intracellular
membranes and implicated in structural roles within the cells in which
they are expressed.

Animals

Fertilized chicken eggs were obtained from Charles River
Laboratories (North Franklin, CT) and incubated in a forced-draft
incubator at 37 °C with saturated humidity at the University of
Florida Whitney Laboratory for Marine Biosciences, St. Augustine, FL.
The care and use of these animals was in accordance with University of
Florida Institutional Animal Care and Use Committee (IACUC) regulations
and the Guide for the Care and Use of Laboratory Animals published by
the Institute for Laboratory Animal Research [6].

Protein extraction

Retinas from twenty E13 chicken embryos were isolated and
homogenized in 10 volumes of lysis buffer (Tris-buffered saline [TBS],
0.1% Triton X-100, 1:1,000 dilution of protease inhibitor cocktail
[product #P-8340; Sigma Chemical Co., St. Louis, MO]) based on the wet
weight of the tissue. The tissue was disrupted via sonication followed
by shaking incubation for 1 h at room temperature. The lysate was
cleared by centrifugation at 10,000 xg for 30 min at 4 °C. The
supernatant was collected and stored at 4 °C.

Affinity purification

The 2M6 antigen was purified using 2M6-specific antibody (University
of Florida Hybridoma Core #HL 1225) [2] coupled to CNBr-activated sepharose 4B,
following the protocol of the manufacturer (Amersham Biosciences,
Piscataway, NJ). Extracted proteins were added to the affinity column
matrix and allowed to incubate on a rotating shaker tray overnight at
4 °C. The unbound fraction was removed from the column and the
resin was washed six times with lysis buffer. The bound fraction was
eluted using 0.2 M glycine, pH 2.5 and neutralized with 1 M Tris, pH
8.0. The eluted fractions with significant absorbances at 280 nm were
pooled and concentrated.

Immunoblotting of proteins

Immunoblotting was performed as described [7]. Eluted proteins
were separated on a NuPAGE 4%–12% Bis-Tris gel in
2-(N-morpholino)ethane sulfonic acid buffer (Invitrogen Corporation,
Carlsbad, CA) and transferred to a nitrocellulose membrane (Osmonics,
Minnetonka, MN). Blots were stained with 0.1% fast green in methanol,
acetic acid, and H2O (5:1:5 based on volume), destained,
documented, and blocked with a 2% solution of nonfat dry milk in TTBS
(TBS containing 0.1% Tween-20) for 1 h at room temperature. After
incubation in blocking buffer, the blots were incubated in 2M6
hybridoma supernatant (1:10 dilution in TTBS; University of Florida
Hybridoma Core) for 1 h at 37 °C. Blots were washed and incubated
in alkaline phosphatase (AP)-conjugated secondary antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:500 for
1 h at 37 °C. The blots were then incubated in AP substrate
(Bio-Rad, Hercules, CA). Protein expression was documented using a
scanner (ScanJet 6100C; Hewlitt Packard, Palo Alto, CA), and the
figures were assembled using Microsoft PowerPoint software (Redmond,
WA).

Mass spectrometry

The affinity-purified sample was cut from the sodium dodecyl sulfate
polyacrylamide gel and submitted for analysis. The gel samples were
washed with digestion buffer (100 mM Tris, 1% reduced Triton
X-100, 10% acetonitrile, pH 8.0), and reduced by incubation in
digestion buffer containing 4.5 mM dithiothreitol for 30 min at
55 °C. Iodoacetic acid (10 mM) was added to the solution, and
the sample was incubated for 30 min at room temperature. The sample was
placed into fresh digestion buffer containing trypsin (1:50 ratio of
trypsin to protein) and incubated overnight at 37 °C. The protein
was extracted from the solution with 0.1% trifluoroacetic acid and 50%
acetonitrile (ACN), dried, and resuspended in loading buffer.

The enzymatically digested samples were injected onto a capillary
trap (LC Packings PepMap300, Dionex, Sunnyvale, CA) and desalted for 5
min with a flow rate of 10 ml/min of 0.1% v/v acetic acid. The
samples were loaded onto an LC Packing® C18 PepMap high-performance
liquid chromatography (HPLC) column. The elution gradient of the HPLC
column started at 3% solvent A, 97% solvent B and finished at 60%
solvent A, 40% solvent B for 60 min for protein identification. Solvent
A consisted of 0.1% v/v acetic acid, 3% v/v ACN, and 96.9% v/v H2O.
Solvent B consisted of 0.1% v/v acetic acid, 96.9% v/v ACN, and 3% v/v H2O.
LC-MS/MS analysis was performed on a hybrid quadrupole time-of-flight
(TOF) mass spectrometer (QSTAR; Applied Biosystems, Framingham, MA).
The focusing potential and ion spray voltage was set to 275 V and 2600
V, respectively. The information-dependent acquisition mode of
operation was employed in which a survey scan from m/z
400–1,200 was acquired, followed by collision-induced dissociation of
the three most intense ions. Survey and MS/MS spectra for each
information-dependent acquisition cycle were accumulated for 1 and 3 s,
respectively.

Tandem mass spectra were extracted by ABI Analyst version 1.1 (Life
Technologies Corporation, Carlsbad, CA). All MS/MS samples were
analyzed using Mascot version 2.0.01 (Matrix Science, London, UK).
Mascot was set up to search the NCBI
database, assuming the digestion enzyme trypsin. Mascot was searched
with a fragment ion mass tolerance of 0.30 Da and a parent ion
tolerance of 0.30 Da. Iodoacetamide derivative of cysteine, deamidation
of asparagine and glutamine, oxidation of methionine, were specified in
Mascot as variable modifications. Scaffold (version Scaffold-01–06–03;
Proteome Software Inc., Portland, OR) was used to validate MS/MS-based
peptide and protein identifications. Peptide identifications were
accepted if they could be established at greater than 95% probability,
as specified by the Protein Prophet algorithm [8]. Protein
identifications are accepted if they can be established at greater than
99% probability and contain at least two identified unique peptides.
Protein probabilities were assigned by the Protein Prophet algorithm [9].

Affinity purification analyses were performed to determine the
identity of the protein recognized by the 2M6 antibody. Previous
reports have indicated that 2M6 is a glial-specific marker expressed
within the developing chick retina [2-4].
Therefore, lysates from E13 chick retina were prepared for use.
Immunoblotting analyses of the eluted fractions obtained revealed the
presence of polypeptides of ~40 kDa, ~46 kDa, and a doublet
of ~80 kDa (Figure 1). These polypeptides
were subjected to in-gel trypsinization so that peptides could be
recovered for MS/MS analyses.

A total of 20 unique peptide sequences were obtained via mass
spectrometry. A search of the NCBI protein database, using the peptide
sequences as queries, revealed that the protein recognized by the 2M6
antibody is identical (95% probability) to a protein previously named
TopAP. The peptide sequences obtained account for 186 of the
359 amino acids within the TopAP molecule, which is 52%
coverage. No other candidate proteins were identified as homologous to
the sequenced peptides. The TopAP (NCBI accession number
gi|642486) sequence is shown with 2M6 peptides underlined (Figure 2).
Hydropathy plot analyses of the sequence for 2M6 suggest that
expression of the polypeptide is membrane associated [5]. The
membrane-associated domain of the molecule, which is found at the
carboxy terminus, is highlighted in Figure 2.

Since it has been reported that the 2M6 antigen is Muller-cell
specific [3,4] and TopAP is thought
to be a neuronal protein [5],
immunohistochemical analyses were performed on sections of intact chick
retina to better localize expression of this protein. An antibody
specific for 5A11/Basigin was used to identify Müller cells of the
chick retina [10,15]. An antibody
specific for neurofilaments [11]
as well as peanut agglutinin, which specifically stains cone outer
sheathes [16],
were used to identify neurons. Figure 3 demonstrates that 2M6
(red) expression overlaps that of 5A11/Basigin (green) and not
neurofilaments (blue). Expression of 5A11/Basigin is observed
throughout the entire Müller glial cells, whereas 2M6 expression is
observed between the ganglion cell layer and the outer limiting
membrane (Figure
3D). Higher magnification views of the expression patterns
of 5A11/Basigin and 2M6 show that while 5A11/Basigin is found on the
plasma membrane of Müller cells, 2M6 is found in the interior of the
cell (Figure
3E–H). Figure 4 demonstrates that 2M6
(red) expression does not overlap that of peanut agglutinin (green) [16] or calbindin [12], which are
neuronal markers in the retina. These findings clearly demonstrate that
2M6 (and hence TopAP) is a Müller cell-specific protein.

Expression of 2M6 was also analyzed throughout the entire retina
since TopAP was reported to have a graded pattern of
expression. E10 chick retinas were isolated, fixed, and embedded in
paraffin for sectioning. Sections were chosen to show the different
stages of differentiation within the chick retina at that age. The
ciliary margin represents an early stage of retinal development with
little cellular differentiation, whereas the fundus represents a late
stage of development in which most cells have differentiated [17]. The region of the
retina at the optic nerve, which represents an intermediate stage of
development, was also examined. These three regions were probed with
antibodies specific for 2M6 (red) and HNK (green). Diffuse-to-specific
staining for 2M6 was observed at the ciliary margin, although HNK
staining is specific throughout this region (Figure 5A–C). Boundary cells, a
population of glia-like cells found at the junction of the neural
retina and the optic nerve [18],
robustly express 2M6 at this age (Figure 5D–F, arrows). In
contrast, HNK is found within the ganglion cell processes leading from
the neural retina (Figure 5D–F, arrowheads).
Analysis of the fundus, which is the most differentiated region of the
chick retina at this age [17],
shows that 2M6 expression is not found in neurons as the signals for
2M6 and HNK do not overlap (Figure 5G–I). These data
indicate that Müller cell-specific 2M6 expression does have a graded
expression pattern in the developing chick retina.

Immunohistochemical analyses were also performed on dissociated
chick retina. Figure
6 shows a cluster of cells that are labeled with the
antibody specific for 2M6 (red), an antibody specific for HNK, a
neuronal cell-surface marker [13]
(green), and DRAQ5, which highlights the cell nuclei (blue). An overlay
of the three channels shows that 2M6 expression does not overlap that
of HNK, which suggests that 2M6 is not a neuronal protein (Figure 6A).
Also, when the Müller cell-derived flat cells adherent to the growth
surface are viewed at high magnification, punctate 2M6 labeling is
evidently associated with endomembranes of specific intracellular
bodies (data not shown). The sequence data suggest that 2M6 is membrane
associated. The immunohistological studies presented herein suggest
that 2M6 is associated with internal membranes and not the plasma
membrane of Müller cells. It has been demonstrated via electron
microscopy that endoplasmic reticulum is widely distributed throughout
Müller cells [19].
Figure 6E
shows a computer-generated “side view” of a 2M6-expressing cell in
which expression is observed throughout the depth of the cell. These
data suggest that 2M6 is associated with Müller cell internal
membranes, such as the endoplasmic reticulum.

Identification of cells in developing tissue is often accomplished
through the use of the molecules expressed by those cells. The 2M6
antigen has been used as a differentiation marker for Müller glial
cells of the vertebrate retina for many years [2-4]. Presently, we
report that the amino acid sequence of the 2M6 antigen has been deduced
by immunoaffinity isolation and subsequent mass spectrometry of
peptides obtained by trypsinization. The peptides match with the amino
acid sequence of TopAP, originally thought to be a neuronal
marker. Immunohistochemical analyses of intact and dissociated chick
retina indicate that 2M6 (TopAP) is indeed expressed by
Müller cells.

The antibody specific for the 2M6 antigen has been used for some
time as a Müller cell-specific marker [2-4],
although the molecular identity of the antigen was not known. The
purpose of this study was to isolate the 2M6 antigen via affinity
purification for mass spectroscopy analyses. The peptides isolated were
compared to known polypeptide sequences and all matched to the protein
known as TopAP (NCBI accession number gi|642486) with at
least 95% probability. The TopAP polypeptide is a member of
the sarcolemmal membrane-associated protein (SLMAP) family of
coiled-coil, tail-anchored, membrane proteins [20]. The SLMAP gene is
rather large, consisting of 24 exons within 122 kb of DNA, located on
human chromosome 3p14.3–21.2 [21-23]. There are several
splice variants produced from the SLMAP gene, each possessing a central
coiled-coil region containing two leucine zipper motifs and a
C-terminal hydrophobic domain responsible for membrane localization of
the polypeptides [21].
The TopAP polypeptide consists of 359 amino acids [5] and is calculated to
be ~40 kDa in molecular mass. The identities of the higher
molecular mass species obtained via affinity purification (~46 and
~80 kDa) are not yet known, but their presence is consistent with
the data reported by Savitt et al. [5] and by others working with members of the
SLMAP and sarcolemmal-associated protein (an outdated name for this
group) family [21].
It is therefore plausible to suggest that the higher molecular mass
species obtained via affinity purification are splice variants of the
2M6 gene found in the retina. SLMAP splice variants have been found in
the sarcolemma T-tubules and sarcoplasmic reticulum of cardiomyocytes
as well as the microtubule-organizing centers (centrosomes) of mouse
fibroblasts [22].
They have been implicated in organizing the excitation–contraction
coupling apparatus and interacting with cardiac myosin in myocytes [23] as well as
interacting with γ-tubulin within centrosomes [22]. Analysis of the
2M6 (TopAP) protein sequence using the SMART program [24,25] revealed the
presence of a prefoldin domain, which is a leucine-zipper motif known
to interact with α- and γ-tubulin [26,27],
as well as a tropomyosin-like domain. Thus, the domain structures
within the 2M6 (TopAP) polypeptide are consistent with
functionalities within Müller cells that parallel those ascribed to
SLMAP proteins in other cell types.

The immunohistochemical analyses presented herein indicate that 2M6
(TopAP) is indeed a glial-specific polypeptide. Both intact
and dissociated chick retina preparations were examined for 2M6
expression and compared to the expression patterns of known
neuron-specific markers. The expression of 2M6 does not localize with
expression of neuronal markers, including neurofilaments [11], calbindin [12], peanut agglutinin
[16], or HNK [13], in intact or
dissociated chick retina. However, expression does correlate with the
Müller cell-specific marker 5A11/Basigin [15]. Savitt et al. [5] did not clearly show that TopAP
was expressed by neurons of the developing chick retina but concluded
this based on the graded expression pattern and recombinant antibody
staining specific for TopAP within the region of the optic
nerve at a time when synapses form in the developing chick retina.
There is a graded expression pattern, as suggested by Savitt et al. [5], in that the fundus,
which is the most developed region of the retina [17], showed robust
Müller cell-specific expression of 2M6, whereas fluorescence
representing 2M6 expression was more diffuse at the ciliary margin (Figure 5).
The data presented herein definitely indicate that neurons within the
developing chick retina do not express 2M6 (TopAP) but that
Müller cells do (Figure 3, Figure 4,
and Figure 6).
This Müller cell-specific expression of 2M6 is confined to the interior
of the cell, as demonstrated in Figure 3, in which 2M6
expression is compared to that of plasma membrane-associated
5A11/Basigin. The 2M6 signal fills the interior of the cell and is
reminiscent of electron micrographs depicting the distribution of
endoplasmic reticulum throughout Müller cells [19].

Based on localization of expression and the conserved motifs found
within the 2M6 polypeptide, it is tempting to speculate about the
function of 2M6 in Müller cells. It has been documented that Ca+2
signaling occurs in Müller cells of the salamander retina such that Ca+2
is released from internal stores and flows from the apical end of the
cell toward the endfoot in a wave-like motion [28]. These Ca+2
waves may provide a second signaling pathway from the outer to inner
neural retina that is independent of neuronal signaling [29]. The 2M6 molecule
may play a role in Ca+2 release from internal stores within
Müller cells as the SLMAP isoform does in myocytes and perhaps
interacts with both the cytoskeleton and the endoplasmic reticulum to
accomplish this feat.